The present report evaluated ultraviolet radiation (UVR) effects on the spectral signature of mycotoxin producing fungus Aspergillus flavus (A. flavus). Ultraviolet radiation has long been used to reduce microbe contamination and to inactivate mold spores. In view of the known effects of UVR on microorganisms, and because certain spectral bands in the signature of some fungi may be in the UV range, it is important to know the maximum acceptable limit of UVR exposure that does not significantly alter the fungal spectral signature and affect detection accuracy. A visible-near-infrared (VNIR) hyperspectral imaging system using focal plane pushbroom scanning for high spatial and spectral resolution imaging was utilized to detect any changes. A. flavus cultures were grown for 5 days and imaged after intermittent or continuous UVR treatment. The intermittent group was treated at 1-minute intervals for 10 minutes, and VNIR images were taken after each UVR treatment. The continuous group was irradiated for 10 minutes and imaged before and after treatment. A control sample group did not undergo UVR treatment, but was also imaged at 1-minute intervals for 10 minutes in the same manner as the intermittent group. Before and after UVR treatment, mean fungal sample reflectance was obtained through spatial subset of the image along with standard deviation and pre- and post-treatment reflectance was compared for each sample. Results show significant difference between the reflectances of treated and control A. flavus cultures after 10 min of UV radiation. Aditionally, the results demonstrate that even lethal doses of UVR do not immediately affect the spectral signature of A. flavus cultures suggesting that the excitation UV light source used in the present experiment may be safe to use with the UV hyperspectral imaging system when exposure time falls below 10 min.